Real-time polymerase chain reaction (PCR)
Specific primers were designed using the Primer-Express program (PE Biosystems) and synthesized by Interactiva (Ulm, Germany). The sequences and necessary concentrations in the PCR reaction are outlined in Table 2. The PCR mixture included 0.625 U AmpliTaq Gold DNA polymerase in the 2× SYBR Green Master Mix (PE Biosystems), the required concentration of specific forward and reverse primers, 10 ng of cDNA template and 0.25 U AmpErase UNG (PE Biosystems) in a 25 μl reaction volume. The quantification relative to the house keeping gene GAPDH was carried out in MicroAmp Optical 96-well reaction plates (PE Biosystems). On each plate a standard curve was generated for both the GAPDH and target PCR reactions by amplifying 5 different known amounts of cDNA derived from total RNA. For each cDNA sample under investigation triplicate reaction wells were set up for both GAPDH and target amplification. The amplification was carried out and analysed in the GeneAmp 5700 Sequence Detection System (PE Biosystems). The efficiency of each PCR was calculated from the slope of the standard curve (E = 10(-1/s) - 1). The abundance of the target relative to GAPDH was calculated as: Xn = (1 + EGAPDH)Ct,gapdh/(1 + Etarget)Ct,target, where Ct is the threshold cycle determined from the amplification curves, and the relative abundances from one quantification were set into relation with one another.
Table 2  The primer sequences and necessary concentrations for the quantification via real-time PCR.
product   forward primer   conc.   reverse primer   conc.
GAPDH  ATCAACGGGAAGCCCATCA  100 nM  GACATACTCAGCACCGGCCT  100 nM
MCP-2  CTTCTCTGGGCTGACAGGGA  300 nM  TCTACGCAGTGCTTCTTTGCC  300 nM
cystatin C  CAAGAAGAGTGGAGCCAGGG  50 nM  GCAGGCAGGTTCTGCACAT  50 nM